PROJECT SUMMARY Histones play a major role in the regulation of transcription across the animal kingdom, and different variants play important roles in a variety of specific tissues and processes. Two recently-described histones, H3.X and H3.Y, are primate-specific and are expressed in human testis and brain, as well as some tumors and cancer cell lines. My preliminary studies have identified these genes as targets of DUX4, a double-homeobox transcription factor that is normally expressed in the germ cells of the testis. When mis-expressed in muscle, DUX4 causes facioscapulohumeral muscular dystrophy (FSHD), a progressive disease for which there are no treatments, and may drive some cancers. Expression of DUX4 drastically alters transcription and leads to de- repression of many genes and sequences throughout the genome that are normally repressed in heterochromatin. H3.X/Y have been validated as histones and shown to facilitate a more open chromatin conformation than H3.3 in vitro, but their in vivo effects in FSHD are as yet unknown. Aim 1 will identify genes that go from off to on and inaccessible chromatin to an open chromatin state with expression of DUX4. It will then determine whether H3.X/Y are preferentially incorporated at these loci as well as examining their effects on expression. This will determine the genome-wide incorporation of H3.X/Y in FSHD and effects on expression of DUX4 targets. Aim 2 will determine the role of H3.X/Y in epigenetic memory of the DUX4 transcriptome. It will extend preliminary studies suggesting that H3.X/Y increase the perdurance of DUX4 target expression as well as examine the effects over time of H3.X/Y on chromatin structure and features of heterochromatin. Additionally, this aim will examine the relationship between cumulative DUX4 toxicity, H3.X/Y, and cell cycle, as mature muscle is postmitotic. Epigenetic memory mediated through histone variants could explain why FSHD pathology is typically restricted to muscle and suggest a mechanism to target for therapy. Additionally, the datasets will be independently useful to determine chromatin changes induced by DUX4 that can be applied to the study of FSHD. This proposal will test the hypotheses that H3.X/Y are incorporated into and facilitate expression of genes transcriptionally activated by DUX4 that are normally repressed by heterochromatin, and provide an epigenetic memory of the DUX4 transcriptional program.